蛛丝蛋白基因在大肠杆菌中表达研究

利用大腹园蛛基因组文库筛选获得一段693 bp基因片段,经分析该段基因处于鞭毛状丝基因重复区域,且其中包含了一个完整的重复框架。通过基因密码子优化,在其3′和5′端分别融合蛋白质亲和层析标签,克隆于pET30LIC表达载体中,在不同的大肠杆菌中进行表达试验。实验结果显示:经过密码子优化,融合目的蛋白基因在BL21(DE3)中得到了高效表达,产量达到25~30mg/L,纯化产物纯度达90%以上。SDS-PAGE和W estern-b lotting检测目的融合蛋白均与预期蛋白大小一致。     

ClC-3 chloride channel prevents apoptosis induced by hydrogen peroxide in basilar artery smooth muscle cells through mitochondria dependent pathway

ClC-3 Cl⁻ channel plays an important role in cell volume regulation and cell cycle. In vascular smooth muscle cells, we have found that ClC-3 was involved in ET-1 induced cell proliferation. The present study was designed to further investigate the role of ClC-3 Cl⁻ channel in H₂O₂-induced apoptosis and its underlying mechanisms in rat basilar arterial smooth muscle cell (BASMCs). By using ClC-3 cDNA and small interference RNA (siRNA) transfection strategy, it was found that overexpression of ClC-3 significantly decreased the apoptotic rate of H₂O₂-treated BASMCs and increased the cell viability, whereas silencing of ClC-3 with siRNA produced opposite effects and increased the apoptotic rate. ClC-3 overexpression decreased cytochrome C release and caspase-3 activation, and increased both the stability of mitochondrial membrane potential and the ratio of Bcl-2/Bax, whereas silencing of ClC-3 produced opposite effect. Furthermore, we demonstrated that overexpression of ClC-3 attenuated, whereas silencing of ClC-3 facilitated, the degradation of LaminA, one of the structural matrix proteins, in BASMCs. Our data suggest that ClC-3 Cl⁻ channel can modulate H₂O₂-induced apoptosis in BASMCs via the intrinsic, mitochondrial pathway.     

A Novel Integrated Method Tor Large-scale Detection,Identification, and Quantification of Widely Targeted Metabolites： Application in the Study of Rice Metabolomics

Liquid chromatography-mass spectrometry （LC-MS）-based metabolomics has been facilitated by the con- struction of MSz spectral tag （MS2T） library from the total scan ESI MS/MS data, and the development of widely targeted metabolomics method using MS/MS data gathered from authentic standards. In this report, a novel strategy called step- wise multiple ion monitoring-enhanced product ions （stepwise MIM-EPI） was developed to construct the MS2T library, in which stepwise MIM was used as survey scans to trigger the acquisition of EPI. A total number of 698 （almost） non- redundant metabolites with MS2 spectra were obtained, of which 135 metabolites were identified/annotated. Integrating the data gathered from our MS2T library and other available multiple reaction monitoring （MRM） information, a widely targeted metabolomics method was developed to quantify 277 metabolites, including some phytohormones. Evaluation of the dehydration responses and natural variations of these metabolites in rice leaf not only suggested the coordinated regulation of abscisic acid （ABA） with metabolites such as serotonin derivative（s）, polyamine conjugates under drought stress, but also revealed some C-glycosylated flavones as the potential markers for the discrimination of indica and japonica rice subspecies. The new MS2T library construction and widely targeted metabolomics strategy could be used as a tool for rice functional genomics.     

全反式维甲酸协同DAPT抑制胶质瘤干细胞的自我更新

前期研究脑表明,脑胶质瘤干细胞（glioma stem cells,GSCs）是胶质瘤发生和发展的重要因素,探索靶向干预GSCs生长有可能成为脑胶质瘤治疗的有效途径之一。该研究旨在阐明两种药物ATRA和Y-分泌酶抑制剂DAPT协同抑制GSCs自我更新的生物学效应。通过用台盼蓝排染法、克隆球形成试验和免疫印迹分析了两种药物的单独使用或联用对GSC样细胞PGCl和PGC2生长、成球能力和自我更新以及干细胞标志物表达的影响。结果发现,单独使用ATRA对PGCl生长有一定的抑制作用,而对PGC2生长几乎没有影响;DAPT对PGCs的生长抑制作用明显强于ATRA。高浓度ATRA（80μmol/L）能诱导PGCs的分化,降低PGCs成球大小,且成球效率降至5％～8％,而正常对照组为32％～35％;同样,DAPT（40μmol／L）也能降低PGCs成球大小,且成球效率降至2％～3％;低浓度ATRA（20μmol／L）和DAPT（5gmol／L）对PGCs自我更新能力和干性没有明显影响,而联合使用后其明显降低PGCs的成球大小,且成球效率降至3％～5％,促进细胞凋亡,并且明显抑制了干细胞标志物Nestin、CDl33、Sox2、Oct4的表达,提高了分化标志物GFAP的表达。该研究证明了低浓度的ATRA和DAPT能协同抑制脑胶质瘤干细胞PGCs的自我更新。研究结果将为脑胶质瘤的临床研究提供实验依据。     

AP-1在腋臭患者发病过程中的作用

目的：腋臭是美容整形外科的常见病,目前发病机制尚不明确,已证实人体大汗腺中的载脂蛋白D（ApoD）在腋臭患者大汗腺中高表达,并且与腋臭的发生密切相关。探明ApoD在大汗腺细胞中的信号转导通路,可以进一步明确其在腋臭发病过程中的作用机制。JNK信号转导通路与多种疾病的发生有关。课题组前期已经做了JNKl对ApoD调控作用的相关研究,证明了在腋臭发病过程中JNK1是通过调控ApoD的转录来上调ApoD的表达。本实验在课题组前期研究基础上,探讨JNKl下游转录因子AP-1是否在JNKl上调ApoD通路中发挥作用。方法：取腋臭志愿者腋区皮肤组织,进行汗腺细胞培养。把汗腺细胞分为5．二氢睾酮处理组、5-二氢睾酮联合姜黄素处理组和空白对照3个组,用姜黄素抑制AP-1的活性,通过Real．timePCR实验方法检测ApoD在姜黄素抑制下的表达变化。结果：在姜黄素的抑制下,ApoD表达明显降低。在体外培养汗腺细胞加入5．二氢睾酮联合姜黄素处理后,ApoD的表达量在mRNA水平低于单独的5-二氢睾酮处理组和正常对照组（P〈0．05）。结论：姜黄素抑制了AP．1的活化导致ApoD的表达降低。在腋臭的发病过程中,JNKl的下游转录因子AP-1对ApoD有明显的上调作用。AP-1可能在JNKl上调ApoD这条通路中扮演了很重要的角色,它可能是JNK1和ApoD的中间转录因子。     

Bradykinin Preconditioning Improves Therapeutic Potential of Human Endothelial Progenitor Cells in Infarcted Myocardium

Objectives

Stem cell preconditioning (PC) is a powerful approach in reducing cell death after transplantation. We hypothesized that PC human endothelial progenitor cells (hEPCs) with bradykinin (BK) enhance cell survival, inhibit apoptosis and repair the infarcted myocardium.

Methods

The hEPCs were preconditioned with or without BK. The hEPCs apoptosis induced by hypoxia along with serum deprivation was determined by annexin V-fluorescein isothiocyanate/ propidium iodide staining. Cleaved caspase-3, Akt and eNOS expressions were determined by Western blots. Caspase-3 activity and vascular endothelial growth factor (VEGF) levels were assessed in hEPCs. For in vivo studies, the survival and cardiomyocytes apoptosis of transplanted hEPCs were assessed using 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodi- carbocyanine,4-chlorobenzenesul-fonate salt labeled hEPCs and TUNEL staining. Infarct size and cardiac function were measured at 10 days after transplantation, and the survival of transplanted hEPCs were visualized using near-infrared optical imaging.

Results

In vitro data showed a marked suppression in cell apoptosis following BK PC. The PC reduced caspase-3 activation, increased the Akt, eNOS phosphorylation and VEGF levels. In vivo data in preconditioned group showed a robust cell anti-apoptosis, reduction in infarct size, and significant improvement in cardiac function. The effects of BK PC were abrogated by the B2 receptor antagonist HOE140, the Akt and eNOS antagonists LY294002 and L-NAME, respectively.

Conclusions

The activation of B2 receptor-dependent PI3K/Akt/eNOS pathway by BK PC promotes VEGF secretion, hEPC survival and inhibits apoptosis, thereby improving cardiac function in vivo. The BK PC hEPC transplantation for stem cell-based therapies is a novel approach that has potential for clinical used.     

Pyrido[2,3-d]pyrimidines: Discovery and preliminary SAR of a novel series of DYRK1B and DYRK1A inhibitors

DYRK1B is a kinase over-expressed in certain cancer cells (including colon, ovarian, pancreatic, etc.). Recent publications have demonstrated inhibition of DYRK1B could be an attractive target for cancer therapy. From a data-mining effort, the team has discovered analogues of pyrido[2,3-d]pyrimidines as potent enantio-selective inhibitors of DYRK1B. Cells treated with a tool compound from this series showed the same cellular effects as down regulation of DYRK1B with siRNA. Such effects are consistent with the proposed mechanism of action. Progress of the SAR study is presented.     

The Helicobacter pylori Protein CagM is Located in the Transmembrane Channel That is Required for CagA Translocation

Helicobacter pylori (H. pylori) is a human gastric pathogen that colonizes the stomach in more than 50 % of the world’s human population. Infection with this bacterium can induce several gastric diseases ranging from gastritis to peptic ulcer and gastric cancer. Virulent H. pylori isolates harboring the cag pathogenicity island (cag PAI), which encodes a Type IV Secretion System (T4SS), form a pilus for the injection of its major virulence protein CagA into gastric cells. Several cag PAI genes have been identified as homologues of T4SS genes from Agrobacterium tumefaciens, while the other members in cag PAI still have no known function. We studied one of such proteins with unknown function, CagM, which was predicted to have a putative N-terminal signal sequence and at least three transmembrane helices. To determine the subcellular localization of CagM, we performed a cell fractionation procedure and produced rabbit anti-CagM polyclonal antibodies for immunoblotting assays. Furthermore, we generated an isogenic ΔcagM mutant to investigate the ability of CagA translocation compared with the wild-type NCTC 11637 strain using GES-1 and MKN-45 cell infection experiments. Our results indicated that CagM was mainly located in the bacterial membrane, partially located in the periplasm, and essential for CagA translocation both in GES-1 and MKN-45 cells, which suggested that CagM was one of the core members of Cag T4SS and localized in the transmembrane channel.